Conventionally, as an analysis method for a trace amount of a substance in a biological specimen, an immunoassay with the use of an antibody-antigen reaction, a DNA hybridization assay or the like has been commonly used. In these analysis methods, a labeling agent for labeling an antibody, an antigen, a DNA, a DNA base derivative, a DNA oligonucleotide or the like needs to be used. As a labeling agent enabling a highly sensitive detection, labeling with a fluorescence, labeling with a radioactive isotope, labeling with an enzyme or the like is commonly used.
Labeling with a radioactive isotope is highly sensitive, however, it has a drawback in that it involves a risk when the radioactive isotope is stored, used and disposed. In addition, labeling with an enzyme has problems in that the molecular weight of an enzyme is high, an enzyme is susceptible to the external environment such as temperature, therefore it is unstable, and its reproducibility is low, and has a drawback in that by binding an enzyme labeling agent to a substance to be labeled, the activities of the enzyme and the labeled substance are decreased.
In addition, as a labeling method with a fluorescence, labeling with an organic fluorescent dye (for example, fluorescein, rhodamine, dansyl chloride or the like) is known. However, it has a drawback in that fluorescence detection of an organic fluorescent dye is disturbed to a large extent by the background noise due to the scattering light of the excitation light or the background noise derived from the fluorescence of other coexisting substances in a sample, whereby it becomes difficult to perform a highly sensitive assay.
As a labeling method with a fluorescence other than this, labeling with a rare earth fluorescent complex is known. A rare earth fluorescent complex has fluorescence characteristics of a long fluorescence lifetime (a rare earth fluorescent complex has a fluorescence lifetime of several ten or several hundred microseconds or more compared with a fluorescence lifetime of several nanoseconds of a common fluorescent substance), a large Stokes shift and a sharp fluorescence peak. By using these characteristics, a time-resolved fluorescence assay method using a rare earth fluorescent complex as a labeling agent has been already developed. Due to these characteristics, by using a time-resolved fluorescence assay, the interference by the background fluorescence with a short lifetime derived from an excitation light or a biological sample can be removed, thereby enabling a highly sensitive assay.
As one of the time-resolved fluorescence assay systems with the use of a rare earth complex as a labeling agent, there is the “DELFIA” (Dissociation-Enhanced Lanthanide Fluoroimmunoassay) developed by PerkinElmer Life Sciences, Inc. (previously, Wallac). This system is a method of performing a fluorescence assay by labeling a protein, a nucleic acid or the like with the use of, as a labeling agent, a complex of isothiocyanatophenyl-EDTA or isothiocyanatophenyl-DTTA (DTTA=diethylenetriamine tetraacetate) with a rare earth ion, adding so-called a fluorescence enhancement solution containing β-diketone-trioctylphosphineoxide (TOPO) and Triton X-100 before measuring fluorescence, releasing a rare earth metal ion from a nonfluorescent complex, and forming a micelle solution of a ternary complex of β-diketone, a rare earth ion and TOPO (E. Soini, T. Lovgren, CRC Crit. Rev. Anal. Chem., 1987, 18, 105-154; E. P. Diamandis, T. K. Christopoulos, Anal. Chem., 1990, 62, 1149A-1157A; I. Hemmila, J. Alloys Compd., 1995, 225, 480-485). However, in this DELFIA system, excess β-diketone and TOPO are present in the measured solution, and if they react with a rare earth metal ion from the environment, they can emit strong fluorescence. Therefore, the system has a major drawback in that it is very susceptible to the contamination of rare earth metal ions. Furthermore, the DELFIA system has drawbacks in that a fluorescence enhancement solution needs to be added, there are lots of measurement steps, and that a solid-phase assay cannot be performed.
As another time-resolved fluorescence assay system with the use of a rare earth complex as a labeling agent, there is the FIAgen system developed by Diamandis et al. in Canada (E. P. Diamandis, Clin. Biochem., 1988, 21, 139-150; E. F. G. Dickson, A. Pollak, E. P. Diamandis, Pharmac. Ther., 1995, 66, 207-235). The FIAgen system is an assay method using a fluorescent europium complex (4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylate(BCPDA)-Eu3+), which can directly label a protein. In this system, there is not a problem of the contamination of europium from the environment, and a solid-phase assay can be performed. However, the fluorescence intensity of the labeling agent in this system is weaker than that in the above-mentioned DELFIA system by at least two orders of magnitude. Therefore, the system has drawbacks in that the detection sensitivity is low, and that a highly sensitive analysis is difficult.
As the other time-resolved fluorescence assay systems with the use of a rare-earth-complex as a labeling agent, there is the TRACE (time resolved amplified cryptate emission) assay system of CIS Bio International in France (G. Mathis, Clin. Chem., 1995, 41, 1391-1397; G. Mathis, J. Clin. Ligand Assay, 1997, 20, 141-147). This system has an advantage in that after reaction in a homogenized solution was finished, a time-resolved fluorescence assay can be continuously performed with the use of a europium fluorescent labeling agent, tris(bipyridine)cryptate-Eu3+, and an organic fluorescent labeling agent, allophycocyanin, as a donor for fluorescence energy transfer and a receptor, and has merits in that a solid phase material is not used and B/F separation or washing operation is not required. However, it has a disadvantage in that the sensitivity is low, therefore, the system cannot be applied to a highly sensitive assay.
To overcome the above-mentioned drawbacks of the time-resolved fluorescence assay method with the use of a rare earth fluorescent complex labeling agent and the systems, the present inventors have already developed chlorosulfonyl quadridentate β-diketone labeling agents that can directly label a protein having an amino group and have investigated an application to a time-resolved fluorescence assay method using these (JP-A-9-241233; JP-A-2000-111480; J. Yuan, G. Wang, K. Majima, K. Matsumoto, Anal. Chem., 2001, 73, 1869-1876; S. Sueda, J. Yuan, K. Matsumoto, Bioconjugate Chem., 2000, 11, 827-831; K. Matsumoto, J. Yuan, G. Wang, H. Kimura, Anal. Biochem., 1999, 276, 81-87; J. Yuan. K. Matsumoto, H. Kimura, Anal. Chem., 1998, 70, 596-601; J. Yuan, G. Wang, K. Matsumoto, H. Kimura, Anal. Biochem., 1997, 254, 283-287).
However, the above-mentioned chlorosulfonyl quadridentate β-diketone labeling agents generally have poor solubility in water. Therefore, it has a drawback in that if it labeled a small biological substance (for example, a nucleic acid base with a low molecular weight having an amino group, or other organic compounds), the solubility of the labeled biological substance is decreased, whereby the substance precipitates from the solution. Also, its chelating ability is not sufficient, therefore, it has a drawback in that the buffer types that can be used are restricted. Accordingly, an application to direct labeling of these substances involves difficulties.
As the information of the prior art documents associated with the invention of this application, there are as follows:    1. JP-A-9-241233    2. JP-A-2000-111480    3. E. Soini, T. Lovgren, CRC Crit. Rev. Anal. Chem., 1987, 18, 105-154;    4. E. P. Diamandis, T. K. Christopoulos, Anal. Chem., 1990, 62, 1149A-1157A    5. I. Hemmila, J. Alloys Compd., 1995, 225, 480-485    6. E. F. G. Dickson, A. Pollak, E. P. Diamandis, Pharmac. Ther., 1995, 66, 207-235    7. E. P. Diamandis, Clin. Biochem., 1988, 21, 139-150    8. G. Mathis, Clin. Chem., 1995, 41, 1391-1397    9. G. Mathis, J. Clin. Ligand Assay, 1997, 20, 141-147    10. J. Yuan, G. Wang, K. Majima, K. Matsumoto, Anal. Chem., 2001, 73, 1869-1876;    11. S. Sueda, J. Yuan, K. Matsumoto, Bioconjugate Chem., 2000, 11, 827-831;    12. K. Matsumoto, J. Yuan, G. Wang, H. Kimura, Anal. Biochem., 1999, 276, 81-87;    13. J. Yuan. K. Matsumoto, H. Kimura, Anal. Chem., 1998, 70, 596-601;    14. J. Yuan, G. Wang, K. Matsumoto, H. Kimura, Anal. Biochem., 1997, 254, 283-287